D uring the studies reported in this thesis I have defined the m inim al set of factors involved in nucleotide excision repair of naked DNA in vitro
by using only recom binant factors for the incision stage, and a m ixture of recom binant factors and purified h u m an proteins for the repair synthesis stage. A m inim um of 26 p o ly p ep tid es are necessary for the full NER reaction: RPA complex (three subunits), XPA (1), XPC-hHR23B (2), ERCC1- XPF (2), XPG (1), TFIIH com plex (m inim um of six sub u n its), PC N A (a hom otrim er), RFC (five subunits), pol 6 /e (two subunits each) and ligase I.
F u rth er proteins m ay be req u ired to deal w ith the ad d itio n al level of com plexity conferred by the chrom atin structure of genom ic DNA relative to the naked DNA substrate. C andidates to deal w ith this higher level of re p a ir co m p lex ity in h u m a n s m ay in c lu d e UV-DDB, th e h u m a n hom ologues of Rad7-Radl6 and hum an MMS19.
UV-DDB protein is the candidate for the p rim ary defect in XP-E cells (Rapic-O trin et al. 1998). UV-DDB has been detected in cell extracts as an activity that preferentially binds dam aged DNA, w ith a particular affinity for
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(6-4) p h o to p ro d u cts in U V -irradiated DNA (Feldberg an d G rossm an 1976; R eardon et al. 1993). This activity has been purified as a complex w ith two subunits of 127 kDa (pl27) and 48 kDa (p48) (Keeney et al. 1993) that w hen microinjected into XP-E cells, lacking UV-DDB activity, substantially correct the NER defect (Keeney et al. 1994; Rapic-Otrin et al. 1998). M utations in p48 su b u n it are associated w ith XP-E in patients lacking this dam age binding activity (Nichols et al. 1996; H w ang et al. 1998). UV-DDB is not a core c o m p o n e n t of th e m a m m a lia n NER m a c h in e ry (th is th e sis a n d (A b o u sse k h ra et al. 1995)), b u t m ay have a specific role in rep air of chrom osom al DNA. B inding stu d ies of this p ro te in to chrom atin, by n u c le ar fractio n atio n , show th a t im m ed iately after U V -irrad iatio n it significantly varies the degree of its association, from a looser to a tighter interaction w ith chrom atin (Otrin et al. 1997). A dditionally, p48 contains a WD m otif th at is fo u n d in p ro tein s in volved in the reo rg an isatio n of chrom atin (Hw ang et al. 1998). An example of such a factor is hum an CAF-1 that is required for reassem bly of chrom atin coupled to nucleotide excision rep air (G aillard et al. 1996; G aillard et al. 1997). Recently, it has also been reported that XP-E cells are defective in global genom e repair of CPDs and that levels of p48 mRNA in norm al prim ary fibroblasts increase after DNA dam age in a p53 dependent m anner (H w ang et al. 1999).
In Saccharomyces cerevisiae, R ad 7 -R ad l6 com plex is req u ire d for
nucleotide excision repair of transcriptionally inactive D N A (W aters et al.
1993; V erhage et al. 1994; W ang et al. 1997). W hen incision interm ediates w ere trapped in vivo in a cdc9ts m utant strain, rad7 and rad.16 m utants were observed to be proficient for N E R -dependent incision in repressed loci like H M L a (Reed et al. 1998). This phenotype led to the pro p o sal of a m odel w hereby R ad7-R adl6 com plex is involved in postin cisio n events d u rin g nucleotide excision repair. These events could be related w ith excision of the dam aged fragm ent. The transcription m achinery could have a role on the excision of the dam aged fragm ent or any other postincision event d uring
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transcription coupled repair and therefore the involvem ent of Rad7-Radl6 w ould only be required for transcriptionally inactive genes. H ow ever, Rad7- R ad l6 has also been reported to have high affinity an d specificity for UV- dam ag ed DNA (G uzder et al. 1997). For this reason, an o th er m odel w as proposed w here this complex is involved in coupling dam age recognition to the assem bly of other NER factors at the site of dam age (G uzder et a l 1998b). This agrees w ith the observation th at R ad l6 m ight also have a role in the repair of the transcribed strand of active genes (Teng et al. 1997).
Sac char om yces cerevisiae M m sl9 has b een iso lated , p u rifie d and
m igrates in SDS-polyacrylamide gels as a 104 kDa peptide (Lauder et a l 1996). M m sl9 appears to be involved in transcription and NER as m m s l9 A cells are deficient in tran scrip tio n -co u p led rep a ir an d global genom e repair (Lombaerts et al. 1997). A ddition of purified M m sl9 does not stim ulate the incision of UV -dam aged DNA, b u t addition of p u rified TFIIH corrects the transcriptional defect also found in these cells. H ow ever, M m sl9 is not a com ponent of TFIIH neither it appears to be associated w ith this factor, suggesting an involvem ent of M m sl9 in a process u p stre a m of TFIIH (Lauder et al. 1996; Lombaerts et al. 1997).
Any of these factors m ight have a role both in the steps leading to the excision of dam age and in regulation of NER in vivo. F urther studies are necessary in order to clarify the roles of these proteins in NER. We currently do n o t know if know ledge of NER in vitro correlates exactly w ith the in
v iv o, cellular situation. It is know n th at NER is sensitive to chrom atin
stru c tu re s because this reaction is in h ib ited in the p resen ce of DNA c o n ta in in g n u c le o so m e s (W ang et al. 1991; S u g asaw a et al. 1993). Accessibility of the repair m achinery to dam aged DNA in chrom atin is still m atter of cu rren t stu d y an d the m echanism by w hich NER locates DNA lesions in ch ro m atin in vivo is n o t know n. M ost p ro b ab ly , chrom atin rem o d e llin g factors like the ones in v o lv ed in b o th rep lica tio n an d transcription can also take p a rt in NER. A dditionally, not only the access of
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NER proteins to chrom atin is necessary, b u t also nucleosom e reassem bly after repair synthesis. A factor also involved in chrom atin rem odelling after rep licatio n , CAF-1, w as sh o w n to be necessary for re p a ir associated chrom atin form ation (Gaillard et al. 1996; G aillard et al. 1997). M ore studies like these are necessary to investigate the in tricate relatio n sh ip betw een NER and chrom atin rem odelling. A nd m ore in vivo studies, as well as the stu d y of all the m ouse knock-out m odels will clarify the subject of in vivo
NER and its implications.